Over the years, there has been considerable interest in the encapsulation or immobilization of living cells, particularly those of microbial origin. See generally, K. Mosbach, Ed., Methods in Enzymology, Vol. 44, Academic Press, New York, 1976; B. J. Abbott, Ann. Rpt. Ferm. Proc., 2: 91 (1980); R. A. Messing, Ann. Rpt. Ferm. Proc., 4: 105 (1980); Shovers, et al. U.S. Pat. No. 3,733,205 (1973). Interest has been extended to the immobilization of plant cells in suspension. P. Brodelius et al., FEBS Letters, 103, 93-97 (1979).
More recently, efforts have been concentrated in processes for encapsulating tissue and individual cells, particularly mammalian cells, so that they remain viable and in a protected state within a membrane which is permeable to the plethora of nutrients and other materials required for normal metabolic functions.
One such technique is described in U.S. Pat. No. 4,391,909 (Lim), wherein tissue cells such as Islet of Langerhans cells are encapsulated within a spherical semipermeable membrane comprising a polysaccharide having acidic groups which have been cross-linked for permanance of the protective membrane. The semipermeable membrane has a selected limit of permeability of no greater than about 200,000 daltons, so that serum proteins and other high molecular weight materials necessary for growth can be sealed with the living cells within the semipermeable membrane, while other, smaller molecular weight metabolites and nutrients can traverse the membrane wall and be interchanged with the outside media. The process therein disclosed comprises suspending the tissue to be encapsulated (and the high molecular weight nutrients) in a physiologically compatible medium containing a water soluble substance that can be made insoluble in water (i.e., gelled), to provide a temporary protective environment for the tissue. The medium containing the tissue is next formed into droplets by forcing the tissue-medium-nutrient suspension through a teflon coated hypodermic syringe, the tip of which is subjected to laminar air flow which acts as an air knife. See also U.S. Pat. No. 4,352,883, wherein the spheres are formed by forcing the materials through a capillary tube into the center of a vortex created by rapidly stirring a solution of Ca++ cation. The medium, e.g. a polysaccharide gel, is temporarily gelled in a generally spherical shape by contact with the calcium solution. Thereafter, these "temporary capsules" are provided with permanant polymeric semipermeable membranes at their outer layer, formed by permanently cross-linking or polymerizing the capsules with polymers containing reactive groups which can react with specific constituents of the polysaccharide.
Thus until the present invention, entrapment in alginate gels alone was considered as only a "temporary" vehicle, around which a permanent membrane could be formed. Generally, following the formation of the permanent membrane, the "temporary" gel was dissolved, so that any cell growth achieved thereafter was not in the presence of the gelled substance.
Such complex prior art processes are not without limitations. For instance, with mammalian cells, although it has been possible to encapsulate viable cells within hardened semipermeable membranes, promotion of growth therein has not been satisfactory. Moreover, cell densities thus far achievable within such membranes has been less than about 10.sup.6 viable cells per milliliter of culture media. Both of these limitations affect the amount and recovery of useful and desirable cell products produced by the entrapped material. The ability to grow cells to higher cell densities within a protected environment (capsule) would provide a means for achieving greater output of desirable cell products.
A further disadvantage of prior art methods of entrapping animal cells is the inability to maintain cell viability at desirable higher cell densities. See P. Brodelius et al., FEBS Letters, supra, where entrapment of mammalian cells resulted in a lack of proliferation of cells and a cell viability of about 10-30% after incubation in tissue culture for one (1) week.